Essentially all eukaryotic cells are polarized, as reflected by the diferent protein and lipid compositions of surface aspects of the cell, as seen, for example, in transporting epithelial cells and neurons. To achieve cell polarity, cells have an organized cytoskeleton that is used by molecular motors to transport organeles and complexes to their appropriate destinations. Whereas the mechanism of motor motility had been extensively studied, much less is known regarding how specific cargos are selected, transported, and delivered, ie the motor delivery cycle. Defects in motors and their cargo receptors, including the highly conserved myosin V family, can cause disease, as is seen in the neurological dysfunction of Griscelli's syndrome. The researcher proposes to use the budding yeast Saccharomyces cerevisiae to study the delivery cycle of Myo2p, the essential myosin V in this organism. The essential function of Myo2p is in the transport of secretory vesicles into the bud for cell growth, but it also transports many organelles into the bud for their segregation during the cell cycle. The study will focus on Mmr1p, a factor that binds the Myo2p cargo-binding domain and has been implicated in mitochondrial inheritance, and that was identified in a genetic screen as functioning with Myo2p in cell growth. Since both mitochondrial inheritance and cell growth are Myo2p-dependent processes, the researcher's hypothesis is that Mmr1p functions directly with Myo2p to contribute to its delivery cycle. Specifically, the researcher first proposes to make conditional mutations in MMR1 and characterize low loss of Mmr1p affects the delivery of secretory vesicles, inheritance of organelles transported by Myo2p, and the delivery cycle of Myo2p. Second, the researcher wil examine the molecular stoichiometry of Mmr1p and Myo2p during the cell cycle, and use live cell imaging to quantitate their association during both the Myo2p delivery cycle, and during the cell cycle. Third, Mmr1p over-expression influences the Myo2p delivery cycle, and it is proposed to use imaging techniques to define which step(s) is affected. Finally, it is likely that Mmr1p associates with other proteins besides Myo2p, so the researcher proposes biochemical and genetic approaches to identify and characterize them. Overall, the work proposed here should provide deep insight into the function of Mmr1p and the delivery cycle of this essential myosin V of budding yeast.